Hot off the Press: Cool Temperature Performance of a Biological Control Agent of the invasive water hyacinth

Wow.. I can’t believe May was my last post.. ugh! Ive been swamped with starting my new postdoc, moving into a new place, and writing an NSF-OCE grant! Anyhow, I am back and will try to be more regular again!

I am excited to announce a new article that is hot off the press! I coauthored this article with Dr. Angelica Reddy (first author) and Dr. Paul Pratt at the USDA, along with researchers from Argentina and Uruguay. You can read it here with free access for 50 days: Article in Biological Control.

This study was in conjunction with some of the work I did as a Delta Science Postdoctoral Fellow to investigate the mechanisms limiting the current biological control of invasive water hyacinth (Eichhornia crassipes) in the Sacramento-San Joaquin River Delta in northern California, USA (hereafter ‘Delta’). Classical biological control uses natural enemies (predators/herbivores, parasitoids and parasites) to control invasive populations of weeds, pests and disease vectors in the introduced range. 
Successful biological control agents can reduce pest populations below threshold levels that cause problems for humans and native species. Once established, biological control can provide a sustainable, long-lasting management option.

In a previous study, my coauthors and I conducted a one-year field survey 34 years after the initial releases of several biological control agents of water hyacinth in the Delta (Hopper et al. 2017). We found that two biological control agents, the herbivorous weevils, N. bruchi and N. eichhorniae (Coleoptera), were still present in the Delta and the associated tributaries. Although N. bruchi was broadly distributed throughout the Delta, N. eichhorniae was only found in the southernmost tributaries. Densities of N. bruchi during the warmer months in the Delta are comparable to densities in other regions with successful control of water hyacinth, but were not high enough year-round to reduce water hyacinth biomass and cover. Thus one idea to improve control is to re-introduce the more rare weevil, N. eichhorniae, in order to increase its abundance and distribution in the Delta and compliment the impacts of the existing weevil populations.

ChillyWeevilOne theory for the difference in the current abundance and distribution between these two weevil species is that the present biotype of N. eichhorniae in the Delta is less cold-tolerant than N. bruchi. Thus, the researchers from the USDA and myself were interested in determining whether a cold-temperature biotype of N. eichhorniae is present. If a cold-tolerant biotype exists, then the goal will be to screen it in the quarantine (host range tests and pathogen screening), access the necessary permits, and then release it into the Delta to improve the performance of the current population of N. eichhorniae and ultimately enhance the control of the invasive water hyacinth in the Delta. 

To achieve these goals, we (Reddy et al. 2019) examined the cool temperature performance and cold tolerance of four populations of the biological control agent, N. eichhorniae. These populations consisted of N. eichhorniae from: the Delta (California: USA), a population within the native range (Uruguay), and two temperate populations (Kubusi River, Stutterheim, South Africa and Jilliby, Australia). The geo-locations of these populations are noted as red markers in the green-highlighted regions on the map.

NE_NB_Map_for BLOGIn this study, we measured life history parameters of these weevil populations under temperatures occurring in the Delta during the cooler seasons (Fall and Winter). These life-history parameters included: Egg survivorship and development, juvenile (larval and pupal) survivorship and development, adult fecundity and adult longevity. I then used these parameters to construct stage-structured matrix models and calculate the intrinsic growth rates, doubling times, generation times and reproductive potential of each of these populations (as I have detailed in a previous blog and linked here).

In summary, Reddy et al. (2018) found that the population from Jilliby, Australia had the highest intrinsic rate of increase under conditions simulating Fall temperatures in the Delta due to the fact this population had the highest fecundity compared to all of the other populations (including the existing population residing in the Delta). Permission is thus being sought to release the Australian population into the Delta to improve the biological control of the invasive water hyacinth.

Please read the published paper for more details! 

And I will be back to update you with much more soon, especially on the results from my study on the population genetics of both of these weevils (N. bruchi and N. eichhorniae), using these same populations pictured above and many others! I finally submitted this manuscript for review.. so stay tuned!

 

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New Post-doc Position at USC!

I am taking a small break from my blog tutorials on using microsatellite markers in population genetic studies to make an exciting announcement: I recently started a new 1-year Post-doc position at the University of Southern California in Dr. Dave Caron’s laboratory (more time pending funding from fellowships)!

USC-Dornsife-Cardinal-Black-on-White-RGBAlthough it is sad that my Delta Science fellowship is over, as it was a wonderful opportunity, I will still be working/writing hard to finish up my publications from this work and I will of course share these with all of you as they are published.phd011817s

In the mean time- I am moving back into marine study systems to examine the diversity and function of protists in the marine phytoplankton community!  Click here to check out the fascinating research in Dave Caron’s lab.  In addition to dabbling in several different ongoing projects in Dave’s lab- I am also very excited about starting up some of my own projects (pending funding) on the abundance, diversity and consequences of parasite-host interactions in the phytoplankton community.  As some of you might already know- I am an extreme parasite enthusiast, and only recently have researchers started to examine the potential abundance and importance of parasites in the marine phytoplankton community!

Recently, researchers in the Tara-Oceans Expedition found that parasitic interactions were the most abundant pattern in the global marine phytoplankton interactome (Lima-Mendez et al. 2015). Results from the V9-18S tag-sequence processing revealed parasite-host associations that included the copepod parasites: Blastodinium (Dinophyceae: Blastodiniaceae), Ellobiopsis (Marine Alveolate Group I: Ellobiopsidae), and Vampyrophrya (Ciliophora: Oligohymenophorea: Foettingeriida) and alveolate parasitoids of dinoflagellates and ciliates (Lima-Mendez et al. 2015). The alveolate parasitoids in particular were recognized for their top-down effects on zooplankton and microphytoplankton (Lima-Mendez et al. 2015).

 

Screenshot 2018-05-12 12.38.19
Figure from: Lima-Mendez G, Faust K, Henry N, et al. (2015) Ocean plankton. Determinants of community structure in the global plankton interactome. Science 348, 1262073.

Parasitoids are parasites that kill their host in order to complete their development (Lafferty and Kuris 2002) and increased abundance of alveolate parasitoids have been linked to declines of dinoflagellate blooms (Coats et al. 1996, Coats 1999, Chambouvet et al. 2008, Mazzillo 2011, Jephcott et al. 2016) and have been shown to regulate their dinoflagellate host populations in laboratory experiments (Noren 2000, Coats and Park 2002). The most researched alveolate parasitoids include several strains of Amoebophrya ceratii (Marine Alveolate Group II: Syndiniales) . These parasitoids have small flagellated infective stages that penetrate and multiply inside the dinoflagellate host cell, and produce numerous infective flagellates after killing and exiting the host (Cachon & Cachon 1987; Jephcott et al. 2016). For example A. ceratii can produce 60-400 new infective dinospores from its host in less than 48 hours (Chambouvet et al. 2008; Mazzillo 2011), and the generalist parasitoid, Parvilucifera sinerae, can produce 170 to > 6000 zoospores per sporangium, depending on the species and size of its host (Garces et al. 2013), with zoospore release within 72 hours of infecting a host (Alacid et al. 2015).

Below for your viewing pleasure is an example of these parasitoids- the life cycle diagram and life-cycle stages from Alacid et al. 2015, and Alacid et al. 2016 (respectively) of the generalist parasitoid Parvilucifera sinerae, in its host dinoflagellates.

So now of course the question you might have is: “why do we need to research these parasites/parasitoids further?” Well, we simply do not  know enough about these amazing parasite-host interactions, and most of our knowledge is currently limited to the photic zone of the ocean, and concentrated on just a few of these parasite species (there are many parasites out there just waiting to be discovered!). For those of you that don’t think ‘not knowing enough’ merits more work- my reply to this is that: mortality rates in the phytoplankton community have an incredible significance regarding the total primary production and biogeochemical processes in the ocean. However, how can we account for the mortality rates in the phytoplankton community and consequences for primary production if we are not accounting for a large % of contribution to mortality due to parasites that have not yet been characterized? And this folks.. is the reason why this research should be funded (aside from the obvious fact that parasites are absolutely fascinating, and the evolution and ecology of parasites can tell us a lot about related free-living species as well (that is another blog topic I will save for the future).

Of course my new Post-doc research in this field is still a bit tentative as it depends on gaining further funding- but in the mean time I am posting some lovely photos of parasites (Euduboscquella spp.) in tintinnid ciliate hosts (Eutintinnus spp.) that I have been finding from some local net tows (marine sampling nets that concentrates organisms of different size classes). So exciting- it is like a treasure hunt every time!

 

References (highly recommended reads also!)

Alacid E, Rene A, Garces E (2015) New insights into the parasitoid Parvilucifera sinerae life cycle: the development and kinetics of infection of a bloom-forming dinoflagellate host. Protist 166, 677-699.

Alacid, E., Park, M. G., Turon, M., Petrou, K. & Garces, E. (2016) A game of Russian roulette for a generalist dinoflagellate parasitoid: host susceptibility is the key to success. Front Microbiol 7, 769.

Cachon J, Cachon M (1987) Parasitic dinoflagellates. In: Biology of dinoflagellates, pp. 571-610. Blackwell, New York.

Coats DW (1999) Parasitic life styles of marine dinoflagellates. Journal of Eukaryotic Microbiology 46, 402-409.

Coats DW, Adam EJ, Gallegos CL, Hedrick S (1996) Parasitism of photosynthetic dinoflagellates in a shallow subestuary of Chesapeake Bay, USA. Aquatic Microbial Ecology 11, 1-9.

Coats DW, Park MG (2002) Parasitism of photosynthetic dinoflagellates by three strains of Amoebophrya (Dinophyta): Parasite survival, infectivity, generation time, and host specificity. Journal of Phycology 38, 520-528.

Chambouvet A, Morin P, Marie D, Guillou L (2008) Control of toxic marine dinoflagellate blooms by serial parasitic killers. Science 322, 1254-1257.

Garces E, Alacid E, Bravo I, Fraga S, Figueroa RI (2013) Parvilucifera sinerae (Alveolata, Myzozoa) is a generalist parasitoid of dinoflagellates. Protist 164, 245-260.

Jephcott TG, Alves-De-Souza C, Gleason FH, et al. (2016) Ecological impacts of parasitic chytrids, syndiniales and perkinsids on populations of marine photosynthetic dinoflagellates. Fungal Ecology 19, 47-58.

Lafferty KD, Kuris AM (2002) Trophic strategies, animal diversity and body size. Trends in Ecology & Evolution 17, 507-513.

Mazzillo FFM (2011) Novel insights on the dynamics and consequence of harmful algal blooms in the California Current System: from parasites as bloom control agents to human toxin exposure PhD dissertation, University of California, Santa Cruz.

Lima-Mendez G, Faust K, Henry N, et al. (2015) Ocean plankton. Determinants of community structure in the global plankton interactome. Science 348, 1262073.

Part II: Tips and Tricks, Multiplex PCR and Workflow of Microsatellites- the cheap way

No this blog is not a belated April fools joke… there really is a method to save thousands of dollars on microsatellite marker multiplex and genotyping! If you are just reading my blog for the first time, this is Part II following up on my last blog: How-to use microsatellites for population genetics, Part I: Study Design, DNA extraction, Microsatellite Marker Design/Outsourcing.

When I first set out to work with microsatellites, I was on a budget and I had never had experience with multiplex PCR, genotyping or fluorescent markers before- so there was definitely an uphill learning curve.

To start (assuming you already have your microsatellite markers- see previous blog for this)- the next step is to order your fluorescent markers to see how things work in multiplex PCR. FYI-Don’t order your Liz Size Standard until you are done troubleshooting and checking things on gels because it expires kinda quick! Plus it ships really fast (at least if you are in the USA). 

What are fluorescent markers you ask? If you look at the microsatellite genotyping peaks above, the different color peaks correspond to the different fluorescent tails that are ‘attached’ to the microsatellite primers. FAM= blue, PET = red, VIC =green, and NED = yellow. This way, you can see the different colors and know what markers they correspond to. The orange peaks are from the 600 Liz size standard which enables you to actually calibrate the size of the peaks. This is very easy if you use a program like Geneious as they have a microsatellite plugin.  I will detail more of the data handling in Part III of this blog series.

Why multiplex PCR? Simple.. it is faster (& cheaper if you troubleshoot things quick).PCR Reaction for Multiplex PCR of Microsatellite Markers

Many folks use multiplex genotyping, where you do singleplex PCR with all the separate microsatellite markers, and then you add for example 4 non-overlapping microsatellite marker amplified products (from your singleplex PCR) into a well together for downstream genotyping (more on that process later). This process saves money, since instead of genotyping a single marker for 1$.. you can genotype 4 markers for 1$! However- singleplex PCR takes forever if you have a lot of samples and markers!!!! If you have 400 individual DNA extracts, and each sample requires 10 markers of genotyping.. this means 4000 PCR REACTIONS- YIKES- that would result in carpal tunnel in a heartbeat!

Multiplex PCR in contrast allows you to add 2-4, (or more) microsatellite markers with fluorescent tails into the PCR mix, making sure that markers with the same fluorescent tails don’t overlap in size (ie- a FAM marker amplified product of 100-250 bp compared to another FAM marker amplified product of ~300-400 bp size range- should be fine to put together in a mixture). Whereas you don’t need to worry as much if they are similar size but have different fluorescent markers (such as FAM (blue) versus NED (yellow)). There are pull-up issues, and inhibition issues.. but that is why you will need to test everything out first anyhow before running your final assays.

As for myself, two papers were key in learning how to streamline microsatellite multiplex and genotyping: Blacket et al. 2012 and Culley et al. 2013. Both studies utilized four universal fluorescent tails (different ones in the different studies)- so that all you need to do is to add the non-fluorescent tail (just the ATCGs) of the corresponding fluorescent tail (the ATCGs + the FAM, VIC, NED or PET fluorescent marker) to your forward primer, and then use pig-tails on your reverse primers (such as GT, GTT, GTTT- depending on your reverse primer). Cullen et al. 2013 has an appendix which actually walks you through every step, including the reaction concentration of each Forward primer +tail, Reverse primer +pig-tail, and Fluorescent marker +tail in the final multiplex mix. I ended up using the four universal tails in Blacket et al. 2012, and then used Culley’s reaction mixes.

Screenshot 2018-04-01 12.55.20
Universal Tails used with PCR fluorophores, from Blacket, M.J., Robin, C., Good, R.T., Lee, S.F., Miller, A.D. 2012. Universal primers for fluorescent labelling of PCR fragments–an efficient and cost-effective approach to genotyping by fluorescence. Molecular ecology resources 12, 456-463.

Below is a great pictorial image of how this works (from Blacket et al. 2012)

Screenshot 2018-04-01 12.54.34
Multiplex PCR with universal tails: Process from Blacket, M.J., Robin, C., Good, R.T., Lee, S.F., Miller, A.D. 2012. Universal primers for fluorescent labelling of PCR fragments–an efficient and cost-effective approach to genotyping by fluorescence. Molecular ecology resources 12, 456-463.

In addition to reading these papers (and their supplementary material) thoroughly, I recommend the following: 

  1. Talk to as many people as you can before you start/ while you are getting started -you always learn fabulous tips and tricks as well as what not to do!
  2. Use the multiplex manager program -this will help you simulate what markers are compatible with each other based on the estimated product sizes, the melting temperatures (Tm) and the specific tails and flourophores you want to use. This will help you think about the different multiplex reactions that you can use.
  3.  Order the Qiagen Multiplex Plus Kit– this will streamline everything! I accidentally ordered the regular multiplex kit.. which is an older version and slower- so I had to stick with it once I got started. However- the Plus version enables you to use a faster PCR protocol! This kit is the same thing as their old ‘Type-it kit”, just better.
  4. Order a set of just the universal tails without the fluorophores attached first, in addition to the forward primers with the universal tails, and the reverse primers with the pig tails. This will let you try running all the multiplex reactions out and test them on a gel to make sure you have everything working before you waste your precious flourophores which are expensive. This is also a cheap thing- 4x $6 max.. 24$ to try out a bunch of stuff before spending the big money is well worth it! By the way  you will order all of your flourophores (reporter dyes) attached to the universal tail or the forward primer (the latter is a more expensive technique) from Thermo Fisher Scientific as they have patents on all of them except for FAM, which you can buy cheaper from Sigma or other companies (IDTdna, Elim BioPharm…etc)
  5. When you have everything working (bands are where you expect them to be, and no large gaps between bands which indicate that the msat markers are targeting multiple regions of the DNA sequence rather than one region)- Then order black, sterile micro centrifuge tubes (this link is just an example- but any sterile brand will work)- this will be what you make all your fluorescent primer ‘party’ mixes in, which will protect the fluorescence from degradation- preventing you from having to order more and save you money and time!
  6. Color code everything -from the tops of your individual fluorescent forward-reverse-fluorescent primer mixes (FRT: Forward-tail +Reverse-pigtail +Fluorophore-Tail) to your tubes with primer party mixes (all four FRT), to your  multiplex pcr reaction mixes and to your excel files for the pcr-plates and genotyping plates.
  7. Be organized when you pipette things onto your pcr-plate, and into your genotyping plate, see below image on how I organize pipetting into a pcr-plate, with the samples in the large tubes being moved in the order that I add things to the plate. I close the lid of each tube and move it to the upper tray after I add it to the plate so I don’t lose my place. I also use my tips from the box in order so that I can look at my tip-box as well to see where I should be. IMG_7752
  8. Talk to the genotyping facility about if they permit a ‘troubleshooting’ run (free-of-charge) so that you can test the amount of final pcr product to add into each genotyping well. I used 0.5 ul pcr product (that had amplified products of four markers) with 11 ul genotyping mix (Liz size standard in Hi-di formamide)

Random Tips/Interesting findings:

  • The Liz size-standard has strict “keep in dark and don’t-freeze’ instructions when it is shipped to you. Be sure to put it in the fridge at 4 degrees and NOT in the freezer! Unfortunately it comes in a styrofoam box w/ ice-packs with no-outside labels instructing to not freeze…and so the mailing department or your lab technician might accidentally put it in the freezer (speaking from personal experience…)- The company (Thermo Fisher Scientific) was very nice in shipping me a replacement because of this issue.  However… due to this occurrence I had the opportunity to test whether or not the Liz -size standard would still work when frozen for 6 hours, and when frozen for 24 hours….Results: The size standard still works great when frozen for 6 hours and for 24 hours ! With that said.. obviously don’t purposely test this, but if it is accidentally frozen- chances are you are still ok!
  • I also found that the Liz size-standard works great and is consistent for at least 2 months beyond its written expiry date…
    • For both of these tests I had unexpired and unfrozen Liz-size standard to compare these tests to. No p-value available.. just my experience 😉
  • As for the genotyping- I used less Liz Size Standard than recommended (and so did everyone that I talked to). My specific reaction mixes were the following: 0.5 ul PCR product + 0.5 ul Liz Size standard, and 10.5 ul Hi-Di Formamide per genotyping reaction well. I know a lot of folks that use 0.5 ul PCR product + 0.2 ul Liz-size standard+ 9.3 ul Hi-Di Formamide.. and they have great success as well. I tried the latter mix  and it worked, but because my pcr products had such high fluorescence (and I was over troubleshooting my primer fluorophore mix concentrations- I decided to  instead increase my size-standard so that I could better separate the noise from the signals). My genotyping mix and final primer ‘party’ mixes result in the initial genotype peaks image of this blog- so you can see what I mean by high sample peaks compared to the size-standard.
  • As for the Hi-Di Formamide- I noticed that this does not have good results when you leave it in the fridge overnight and try to use it the next day for a second genotyping plate. However- I had good results with using Hi-Di Formamide that underwent 1-3 freeze-thaw cycles. Thus my advice is that you never leave it at room temperature or in the fridge if you have extra, but also to avoid too many freeze-thaws.
  • Additionally- Im sure you will find this out- but NEVER freeze your pcr-products after the multiplex pcr with the flourescent markers, or after you add the liz-size standard and hi-di formamide. If you can’t get your samples to the genotyping facility right away, then be sure to do a quick denature (95 degrees for 5 min) post combining the pcr product w/ the Liz size standard and hi-di formamide and then just keep in the fridge (and in the dark!) until the next day or two.

References

Blacket, M.J., Robin, C., Good, R.T., Lee, S.F., Miller, A.D. 2012. Universal primers for fluorescent labelling of PCR fragments–an efficient and cost-effective approach to genotyping by fluorescence. Molecular ecology resources 12, 456-463.

Culley, T.M., Stamper, T.I., Stokes, R.L., Brzyski, J.R., Hardiman, N.A., Klooster, M.R., Merritt, B.J. 2013. An efficient technique for primer development and application that integrates fluorescent labeling and multiplex PCR. Appl Plant Sci 1.

 

 

 

 

 

 

 

 

How-to use microsatellites for population genetics, Part I: Study Design, DNA extraction, Microsatellite Marker Design/Outsourcing

So… you want to use microsatellite markers to assess the genetic variation and population structure of your focal study organism? Well if you are anything like me two years ago.. then you have no idea where to start. Otherwise- congratulations if you are already an expert- in which case you probably don’t need to read on 🙂

SeeHearSpeak
“See No Weevil, Hear No Weevil, Speak No Weevil”                                                                          Illustration by Jacki Whisenant, contracted by Julie Hopper. Copyright 2017.

Two years ago, I was just like you (and these weevils above), and felt a bit overwhelmed and lost in undertaking the large task of designing microsatellite markers and genotyping these markers for the two weevils species (Neochetina bruchi and N. eichhorniae) that I have discussed in previous posts. 

Very briefly to recap on my work:  these two weevil species are used all over the world for the biological control of the invasive water hyacinth, including the Sacramento-San Joaquin River Delta, California. They have had variable success, with notable reduction of biomass and cover of water hyacinth in warmer climates compared to more temperate climates such as the Delta. Although temperature plays a large role in their success, I am also investigating the role of genetic variation and particularly whether there is lower genetic diversity and heterozygosity in the Delta compared to the native origin of these weevils (Uruguay and Argentina).

In Part I- (this blog), I will detail the how-to’s of sampling design and strategy, and the development of (or outsourcing) microsatellite markers.

In Part II- (next blog) I will discuss how to make your final microsatellite marker selections, and the workflow of multiplex PCR and genotyping.

In Part III- (come back in a month!) I will detail how to analyze the data with various R-packages and other computer programs, and how to format the data files correctly for these programs.

On this note, please research your study system thoroughly, as every organism is different and may require different sampling strategies and methods than I detail here for two diploid beetle species (Insecta). Additionally.. my overview below on Part I- is very brief and I definitely skip small steps to be succinct. Also my suggestions are not the only way to do things and below this blog, I post links to several other great resources. Lastly- This work is currently in prep for publication and I will post an update again after publication.


Part I: 

 

Figure from: Grunwald et al. 2017, Phytopathology
Figure from: Grunwald, N.J., Everhart, S.E., Knaus, B.J., Kamvar, Z.N. 2017. Best Practices for Population Genetic Analyses. Phytopathology 107, 1000-1010.

Sampling Design and Strategy:

First before you start sampling or ordering primers- make sure that you have a solid study question with a testable hypothesis, and a good study framework.

Next: all of the power in your genetic analyses (aka, accuracy and ability to detect differentiation among populations, etc.) depend on: 1) your sample quality (aka DNA quality), the number of samples (replicates) per treatment or location, 2) the number of high quality microsatellite markers (e.g.quality relating to two important characteristics: markers are polymorphic -having 2 or more alleles per locus-with more being better, and the markers lack true null alleles), 3) the robustness of your PCR  – whether the PCR conditions are truly suitable for your markers, and whether they can result in reproducible data, 4) the assumptions of the data and 5) the choice of statistical tests and whether the tests are truly suitable for the data.

I will cover the latter (regarding statistical tests) in a future blog, but for today I would like to focus on the ideal # of samples and the # of polymorphic markers. There has been debate about how many samples and how many markers are necessary for robust studies, and if you study an endangered species -sometimes you just have to work with what you got!

In a perfect world– you will want to make up for what you lack in samples with microsatellite markers (loci) and vice versa. So if you have a lower end of replicates, then you will want a higher number of microsatellite markers (# of loci, and more important is to have polymorphic loci with 2 or more alleles/locus) to test for each individual (replicate), and again vice-versa. There are a couple great papers that discuss sampling strategies and study design that you should definitely check out, particularly the one noted in the figure above (Grunwald et al. 2017), as well as Hale et al. 2012 which states that 25-30 individuals per population should be sufficient to accurately estimate allele frequencies given population (with some caveats). Caveats being that obviously, 25-30 individuals per population would likely NOT be enough if you only have four microsatellite markers, particularly if these markers are not polymorphic or very variable (variability referencing to the # of alleles per locus- the more the better!).. so keep this in mind. In general, with that many samples- 10-15 polymorphic markers should be fine (although the more the better), but again this depends on your study question and study system. Also, more samples might be necessary if you are interested in population differentiation (population genetic structure). In fact, in a landscape genetics study, Landguth et al. 2012 demonstrated that increasing the number of loci (and particularly having more variable loci) is more likely to increase the power of population genetic inferences compared to increasing the number of individuals.

You can also test your samples with genotype accumulation curves to see if you have captured the majority of genetic variation (I used the poppr package in R for this and will discuss more on poppr and its primer in Part III of this blog series).

With that said.. If I would have known 1 year ago what I know now…. I would have asked for folks around the world to collect more weevils for me, and I would have extracted more DNA!  Just remember.. not all of your DNA extractions are going to end up working out..due to various human error and/or preservation issues. Thus its always good to add at least 10-20 more samples than you think you need!

map_with_labels_pop_gen
Sampling locations of Neochetina bruchi and N. eichhorniae individuals that I used for the focal population genetics study (Hopper et al. In Prep). Thanks to all those who sent me weevils!

Designing or Outsourcing Microsatellite Marker Design: 

  • Marker Outsource Options: I want to first be upfront in that I actually ended up outsourcing this component of my study as I was going through a tough time and taking care of my dad who had metastatic cancer via at-home hospice care in Columbus, Ohio for two months. Needless to say- I was working remotely then, which made the decision to outsource this part of the lab work an easy decision. I researched a lot of outsource options and in the end I went with the cheaper and most recommended option by several colleagues- the Savannah River Ecology Lab at the University of Georgia. In the end I have mixed opinions on their work and please email me if you would like more info and I will detail the ups and downs.
  • Brief Workflow for designing microsatellite markers: 
    1. First! Check the literature to make sure microsatellite markers have not already been developed for your species or a sister species (the latter of which will sometimes work). Using previously developed markers is obviously the easiest and cheapest route!
    2. If the markers have not already been developed: Obtain high quality and high molecular weight DNA Extractions. I love doing 5% Chelex DNA extractions, but the resulting DNA can be full of PCR inhibitors- so I always use the second half of the DNAeasy kit to purify and clean up my DNA samples. You can also buy replacement spin columns for these kits way cheaper from Epoch Life Science. Then quantify them on a nano-drop or a similar DNA quantification instrument and additionally run them on a gel to make sure that you have ≥100 uL of ≥50 ng/uL of >10kb DNA per sample.
    3. Send to a sequencing facility (Illumina with paired ends >150bp preferred)
    4. Clean up sequences/fix Errors and Run a program called “Pal_finder”, or use a similar program. Pal_finder can analyze 454 or paired-end Illumina sequences ( ~150bp from each end).  This program sends possible primers to Primer3 for primer design and searches for how often each primer and primer pair occur.

    5. Filter the resulting data set by only including: a) sequences for which primers can be designed (e.g. enough flanking sequence) and b) primer pairs that occurred 1-3 times. Then, sort by motif length (di, tri, tetra, etc.) to quickly find tri or tetra nucleotide repeats and look to see if the motif was found in both directions of the sequence (which can be bad as they typically end up being smaller PCR products, but this depends on your goals). Finally, order a bunch of the primers that look promising-say 48 primer pairs to start, and test them out on a subset of 24 individuals, with an equal distribution of these individuals across all your study locations, or select individuals that you think will have a lot of variation. See Initial PCR testing in the next Blog. 

To be continued…

References

Grunwald, N.J., Everhart, S.E., Knaus, B.J., Kamvar, Z.N. 2017. Best Practices for Population Genetic Analyses. Phytopathology 107, 1000-1010.

Hale, M.L., Burg, T.M., Steeves, T.E. 2012. Sampling for microsatellite-based population genetic studies: 25 to 30 individuals per population is enough to accurately estimate allele frequencies. PloS one 7, e45170.

Landguth, E.L., Fedy, B.C., Oyler-McCance, S.J., Garey, A.L., Emel, S.L., Mumma, M., Wagner, H.H., Fortin, M.-J., Cushman, S.A. 2012. Effects of sample size, number of markers, and allelic richness on the detection of spatial genetic pattern. Molecular ecology resources 12, 276-284.

Helpful Resources on Getting Started for Part I

Lecture on Intro to Microsatellites

Part II Stage Structured Matrix Models: Measuring the Intrinsic Rate of Increase in PopBio, R

Woohoo- I am officially done with my lab work for the population genetics study on the two biological control agents (Neochetina bruchi and N. eichhorniae) of the invasive water hyacinth. I will post a blog updating you on the how-to’s and my results soon! Finally – I am done driving back and forth to the Bay Area- and currently just working on data analysis and writing up everything here in LA. Stay tuned!

phd053104s

In the mean time…

As I promised, here is the Part II to my recent blog:  How-To: Stage-Structured Matrix Models. 

In this last blog, I discussed the importance of stage-structured matrix models in calculating the intrinsic rate of increase of organisms with developmental stages (such as the weevils!) and detailed how to construct a stage-structured matrix models in excel. Again here is that file: Julies_tutorial_example_matrix_for_popbio

So now that you have your matrix.. what do you do next?

1st: Convert your matrix into a csv file such as the one I created below based on the excel file above. Just remember- don’t include the headers or row names. I am unable to upload, so I am pasting a picture of the matrix in excel below. Screenshot 2018-01-29 11.24.16

2nd: Save this file as a csv to your working directory that you use in the R statistical program. If you haven’t used R before, then go to the R website to download the program, and refer to the below links on how to set up R, your working directory, and how to import files:

1) http://sphweb.bumc.bu.edu/otlt/MPH-Modules/BS/R/R1_GettingStarted/R1_GettingStarted8.html

2) http://www.r-tutor.com/r-introduction/data-frame/data-import

Also I highly recommend downloading R studio before you start as well -makes everything a lot easier- download R studio here

3rd: Install the ‘popbio’ package. You can read about this package here.

4th: Run the below code!

#below tutorial on getting a matrix model into R and analyzing with pop bio
library(popbio)
library(popdemo)
help(popbio) #this provides more information on this package
tutorial.mm=read.table(file=”tutorial_matrix_pop_bio.csv”, sep=”,”, header=FALSE)
tutorial.mm
tutorial.L=lambda(tutorial.mm)
tutorial.L
intrinsic=log(tutorial.L)
intrinsic

#You should obtain an intrinsic rate of increase of  0.0217994

5th: If you want to analyze the stable stages, reproductive values, net reproductive rate , generation time,and conduct an eigen analysis…. Then you will also need to do the following: 

#below to get more information:
###############NEED TO FIRST LIST STAGES
stages<-c(“egg”,”1st_2nd_larvae”,”3rd_larvae”, “pupae”,”pre_rep”, “adult”)
colnames(tutorial.mm)<-stages
rownames(tutorial.mm)<-stages
tutorial.matrix=as.matrix(tutorial.mm)
tutorial.matrix
stable.stage(tutorial.matrix)
reproductive.value(tutorial.matrix)
eigen.analysis(tutorial.matrix)
fundamental.matrix(tutorial.matrix)
net.reproductive.rate(tutorial.matrix)
generation.time(tutorial.matrix)

#Congratulations! You did it!

 

 

Work featured in Estuary News, December Issue

Currently I am still plowing away in the Bay Area at the last samples for the population genetics study on the weevil biocontrol agents that I will detail in a later post. I have a new-found carpal tunnel from all of the pipetting.. lol. I try to even it out with constant rolling on my favorite foam roller!

In the mean time-I am thrilled to share the current December issue of SF Estuary News with you, as they recently featured my work on exploring the mechanisms limiting the current biological control of the invasive water hyacinth in the Sacramento-San Joaquin River Delta in California. Check out the profile here: SF Estuary December Issue Profile. And here is the link to the pdf file

Thank you again to the Delta Science Council and California Sea Grant for  funding this work through the Delta Science Fellowship!

Screenshot 2017-12-15 10.56.27

 

 

How-To: Stage-Structured Matrix Models

Happy Thanksgiving everyone!

So this week I constructed several stage-structured matrix models- aka Lefkovitch models- to estimate the finite and intrinsic rate of increase of the weevils Neochetina eichhorniae and N. bruchi under laboratory simulated Fall and Winter conditions in the Sacramento-San Joaquin River Delta. This work is in conjunction with a postdoctoral researcher- Angelica Reddy and Paul Pratt’s laboratory at the USDA to test the temperature performance of these biological control agents.

As you know, if insects are adapted to warm weather- they don’t perform very well in colder temperatures and this can be very applicable to biological control agents (such as the two Neochetina weevil species) that are brought from their tropical origins to colder regions to control invasive species.  Below is a cute cartoon I had a scientific illustrator Jacki Whisenant draw for me, and for a new children’s book we are writing…stay tuned!

ChillyWeevil
Copyright 2017, illustration by Jacki Whisenant and made for Julie Hopper

Because we are working in the laboratory on these two species we are able to gather a lot of life history parameters of the weevils undergoing Fall and Winter conditions. These parameters include: development time and survivorship of the different insect stages (egg, III instars of larva, pupa, pre-reproductive adult and reproductive adult), as well as the emerging sex-ratios, and longevity and daily and lifetime fecundity of the reproductive female adults.

From these parameters we can conduct several different analyses to approximate the finite rate of increase, intrinsic rate of increase, generation time, doubling time and net reproductive rate of a species to understand more about their potential population growth rates (which of course is important for biological control).

My favorite way to approximate these population growth parameters for insects is to use a stage-structured matrix model (Lefkovitch model). There are other methods you can use as well- but I won’t go into that here. If you would like to read more see the citations at the end of this blog.

Instead, I will provide a how-to tutorial since while I was working on these matrix models as a graduate student- I realized there is a lack of tutorials on the web on how to construct these models in an intuitive manner. I got lucky  as both my PhD adviser and one of my lab mates (whom had already done the research on stage-structured matrix models) helped me understand how to construct and interpret the models. In the name of paying it forward- I am attaching here an excel worksheet that has all of the calculations and formulas that demonstrate how to construct these stage-structured matrix models (see link). 

Julies_tutorial_example_matrix_for_popbio

In my next blog- I will detail how to use this resulting matrix and input it into the package popbio (Stubben and Milligan 2007) for calculation of finite rate of increase (lambda), intrinsic rate of increase (r), doubling time, generation time, net reproductive rate and much more!

Disclaimer- this is for insect stage-structured matrix models only as calculations differ for plants and vertebrates typically.

Here are some of the calculations that are built into the excel formulas: 

Screenshot 2017-11-22 12.53.45

Below is another screenshot of the file: 

Screenshot 2017-11-22 12.57.42

Below is a diagram from the famous study on Loggerhead sea turtles that explains the flow of this matrix better. However be aware that the matrix above and in the attached excel sheet-calculates gamma as 1/duration which is very different than the famous example on turtles (below), and from any matrix with plants- mainly due to life history differences among plant, invertebrates and vertebrates.

Screenshot 2017-11-22 13.10.04.png
A Stage-Based Population Model for Loggerhead Sea Turtles and Implications for Conservation Author(s): Deborah T. Crouse, Larry B. Crowder, Hal Caswell Source: Ecology, Vol. 68, No. 5, (Oct., 1987), pp. 1412-1423

References 

Caswell H (2001) Matrix Population Models: Construction, Analysis, and Interpretation. Sinauer Associates, Sunderland


Resources:

Awesome powerpoint by Chris Free at Rutgers 

Another awesome tutorial on primarily the Leslie matrix from UCSC

 

 

Invasive Weed Alert! Alligatorweed in the Delta and Suisun Marsh, California

I just received word from Louise Conrad (Department of Water Resources) that there have been recent sightings of Alligatorweed (Alternanthera philoxeroides) in both Suisun Marsh and the Tower Bridge marina in the east Delta. This is a new, noxious, weed to the Sacramento-San Joaquin River Delta system and we should all be on the look out (see photo above and below).

This invasive weed, native to South America, forms floating mats but it is rooted in sediment and has submerged, floating, and emergent forms. This invasive weed can survive a wide range of environmental conditions making it particularly threatening to both aquatic and terrestrial ecosystems.    For more information on this weed- please see: http://www.sms.si.edu/irlspec/alternanthera_philoxeroides.htm

In California, currently this weed has been documented near Grizzly Island in August, 2017 and from two other sites further up the Montezuma Slough channel in September.

The introduction point is unknown, but it is clear that this weed is in the Sacramento River and has now moved into Suisun Marsh.  Other naturalized locations can be expected.  A rapid response to the alligator weed in Suisun is warranted before this invasion compromises planned tidal wetland restoration projects.

On a related note, the invasive yellow flag iris (Iris pseudacorus), and  Ludwigia hexapetala  (Uruguayan primrose-wllow) are also spreading in these same areas and should be reported as well if found (see details for contact info below).

 

Alternanthera_philoxeroides_discoverlife.org
High densities of Alligatorweed (Alternanthera philoxeroides), forming dense mats. Also called “Pig Weed”. Photo credit: http://www.discoverlife.org

KEY ACTION POINTS: If you find new populations of alligatorweed (or the other weeds mentioned above) please take photos, GPS points (can be via your smart phone), and a voucher specimen (if possible) to send/email to the State Taxonomist, Genevieve Walden, at CDFA Genevieve.Walden@cdfa.ca.gov

 

It is crucial to notify Genevieve Walden as we need to document the extent of the problem. If we are not able to control the spread of this weed immediately it will result in similar issues and problems resulting from Brazilian Waterweed, Water Hyacinth and Water Primrose.

What happens if this weed spreads you ask?


Biological control is a possibility, and in fact, one of the biological control agents, the alligatorweed flea beetleAgasicles hygrophila, has the distinction of being the first biocontrol insect released in the U.S. in order to combat an invasive aquatic weed!

ahygrophila1
Agasicles hygrophila, Photo Credit: http://www.sms.si.edu/

Overall, management impacts on alligator weed by the alligatorweed flea beetle have resulted in a dramatic decrease in the amount of infested aquatic habitat since the insect was first released.

However, the most effective and easiest solution to combating alligatorweed in the California Delta and Suisun Marsh regions is to prevent it from spreading in the first place!

 

 

 

 

How to Get into Grad School: Part I

The past month has been crazy again (or maybe this is just how I start all my blogs?).

My husband and I made the big move to Los Angeles as he started his PhD program at the University of Southern California (USC). In-between moving,  I’ve been driving back and forth between LA and the Bay Area to finish up my lab work for my current postdoc position so that I can get all of my data and remotely work the rest of the year in LA.

Screenshot 2017-09-29 14.38.51.png

When Im not in the Bay Area living the couch surfing and pipetting life style, I’ve been surveying my favorite coffee shops in the West-Adams area (so far Nature’s Brew and Cafe Ignatius are the winners!) and submitting postdoctoral research proposals for several fellowships as well as applying for jobs.. real big time professor jobs!!! Fingers crossed that something comes out of all of this!

However, while I was working on all of my research and teaching statements, I realized that it might be useful if I posted a blog on the how-to’s of graduate school.. since I have now successfully put that behind me.

For Part I, I will focus on just the ‘admissions’ aspect of graduate school.

I will have a Part II later on with details on how to succeed and survival it all.

Disclaimer: This advice mainly pertains to PhD programs and STEM fields, and may differ slightly if you are applying for a masters degree or other programs (non-STEM).

61BeeTO-NQL._AC_UL130_

Part I: How to Get Into Graduate School


A: What to do as an undergraduate to prepare for applying for Graduate School?

EXPERIENCE EXPERIENCE EXPERIENCE! Get involved in whatever research opportunities that you can. Don’t be too picky if it is your first research experience, since opportunities lead to more opportunities and every opportunity will give you a little bit more insight on what you are passionate about. Furthermore.. every research project should have the same fundamental scientific process (or it should anyhow) and so you will learn about science and how to conduct research by taking advantage of research opportunities that are available. After you have a little bit of experience, try to conduct your own independent projects either in a class setting, under the mentorship of a professor, postdoc or graduate student and/or apply for undergraduate research fellowships.                      

MAKE CONNECTIONS WITH PROFESSORS & RESEARCHERS- In addition to going to office hours and talking with researchers at your University, research experience will also help you establish real connections to professors, postdocs, and graduate students. Connections are critical for meaningful letters of recommendation, as well as getting advice on your next steps, regarding what research programs, advisers and universities are a good fit for YOU!

GRADES AND GRE SCORES…do they matter? 

phd120902s          Short answer: Yes, but don’t kill yourself over it. Long answer: As long as you don’t have horrible grades and GRE scores (e.g. B’s are fine, and being in the average range is fine), your research experience and personal character will be just as important to the graduate admissions committee.  Regardless, since you still have to take the GRE for most university programs, start studying at least 6 months in advance of your test date.

B: How the Heck do you Choose a Graduate Program and/or Adviser?

  1. Figure out what you are passionate about!!! Think about what research projects and what types of courses inspired you? The ones you liked studying for? Maybe this is a clue to what you are passionate about!Hopefully your research experience and courses as an undergraduate can give you a head start on this critical component. If you need more help on finding out what really moves you- go to your school library and sit down with some various peer-reviewed research journals (Science, Nature, Ecology, PNAS, etc) and browse through them until a topic hits your interest. You can also browse an Internet research browser such as ISI web of science, but this is a bit harder if you don’t know where to start!
  2. Do you have any location limitations for graduate school? Write out a list of places you would be willing to live (or not to live in).
  3. Browse the faculty members and their research profiles at prospective Universities that you would like to go to.
    1. Look at their CVs, and the publications they have produced
    2. Look at how many graduate students they have mentored and how many students have graduated.
    3. Read several publications and see what they are currently working on.
    4. If any of their research interests you: EMAIL THE PROFESSOR!!
  4. Email the Professor of interest and think about Applying to the University 

The email should go something like this: (substitute specific and personal items for XYZ)

 Dear Professor XYZ,

            I am very interested in your research on XYZ because I have experience with xyz ….. I read your article entitled “XYZ” and was particularly intrigued in the fact that… ……I would love to focus on this research area for my PhD at the University of XYZ in your laboratory. Would you have any time to discuss the potential for working in your laboratory? If you do not have available funding or positions, would you be willing to work with me to apply for funding or able to recommend another laboratory in this field of research?

I have also attached my resume for your review. 

Thank you for your time, XYZ

  1. Email a ton of other professors as well (don’t put all your eggs in one basket!)
  2. Look at other research profiles at various Universities
  3. Think about possible grant-writing opportunities and come up with a rough-draft proposal if you think you need to take this direction.
  4. Email professors again (in two weeks) if they haven’t responded.
  5. Don’t take a lack of response or rejections personal
  6. Schedule interviews (phone, Skype or In-Person) for yourself with the professors you emailed if they respond positively. I actually scheduled my own meetings with professors I wanted to work with at UC Berkeley since I was in town for a week coincidentally, and I think that helped! 

5. Have an Interview?! Dress nicely (even in on the phone!) be on time, and come prepared (e.g. read all the papers you can in the area of research you plan on discussing with the Professor.. particularly the articles the professor authored!). Provide your thoughts on some of the theory in the field and ask lots of questions.

C: How do you apply to a graduate program? and When?

****Establish Connections****Hopefully from the above, you can see how important it is to establish a connection with one or more professors from the program and university of your interest.

PUT A LOT OF EFFORT INTO YOUR PERSONAL STATEMENT and/or STATEMENT OF PURPOSE: You should be passionate about something and this should be very apparent in your  statements. Be careful and stay honest. Don’t say you have experience with things that you don’t, and don’t propose an area of research that you are not interested in. Also, if you are interested in Ecology, Evolution and/or Environmental Science- it is beneficial to list specific faculty that you have already made contact with, and state how your interests align with their research. For MCB/Chemistry and maybe some other departments- my understanding is that they mainly do lab rotations, and so you would want to list several faculty at least.

For your personal statement, let the admissions committee see who you are as a person, and how this has contributed to your passion in the field that you are applying to. What sets you apart from others? Bring in your personal story- how you grew up, your cultural and economic background (the latter only if you had disadvantage and you overcame it).

These statements are tremendously important, so proof read your statements (grammar/spellcheck!). Send your statements out to former grad students, postdocs and faculty that you worked with during your undergraduate years for more feedback. Don’t forget to tailor the statements to each of the schools you are applying to and be careful if you are copying and pasting to not list a different school for the current school you are submitting your essay to.

Do not underestimate the power of these statements. 

Timeline: You should be studying for the GRE’s around June-August, Take the GRE early in case you need to retake it, make contact with prospective advisers/labs around August-October, working on your statements in November/December and applying for Schools in December/January. * Note this is mainly from my experience with schools in the USA and may vary across the globe.

D: Congrats- you got in to multiple places! How do you choose?

phd0406s

If you get into multiple places- make sure you visit each University and each prospective lab that you would want to work in. For the University level: Do you like the location? e.g- would you be happy with  climate and surrounding area? Does it suit your personal life? e.g. If you have a partner- how do they feel about relocating to that area? How does the University compare to your other options in terms of reputation and funding?

For the Department Level: What is the funding situation like? How much money will you make and is it enough to live off of in the surrounding area? Do you need to teach? If so how much would you teach? Teaching is great,  but you don’t want it to interfere so much with your research that it takes you 10 years to graduate! Will you have health insurance? How are the benefits compared to your other options? Also- how is the social culture in the department? Do faculty, postdocs and graduate students all work together well? Are there at least some social events that the Department hosts? Do graduate students seem happy? What are the career support options? (eg. do they have a career center or folks that can help you with job applications when the time comes?)

Lastly and perhaps the most important:  At the Lab/Adviser level:

What is the adviser like? Is he/she hands-on or hands-off? Which of these suits you best? Are there lab meetings or does the professor (adviser) schedule one-on-one meetings with the students? How available is the professor? e.g. – Are they chair of the Department? If so then just keep in mind they might be more busy than other professors and might be more hands-off while they have that position (doesn’t always hold true, but something to ask them about at least if they do have a demanding position).

Are the graduate students in the lab relatively happy and productive? Have the graduate students published papers in a timely manner? Have former graduate students secured relevant jobs (government/academia/biotech)? Does everyone in the lab seem to work well together?

What are the laboratory facilities like? Do you have access to all of the equipment/materials that you need (either in the prospective lab or in willing labs nearby?) Are things relatively organized, clean and safe?

Does the research still interest you when compared to your other options? Lastly- is the research in an area of high funding? This might be important if the Department can’t pull together enough funds for your research or stipend.

Weigh all of your pros and cons and then go with your gut feeling! Good Luck!

 

 

 

Society for Invertebrate Pathology, 2017

This week has been intense and fun, as yesterday was the last day of the five-day SIP Conference  (Society for Invertebrate Pathology) in San Diego California!

This society and conference focuses on invertebrate pathogens, with seven different divisions, and works in conjunction with the Journal of Invertebrate Pathology.

Divisions
The seven divisions that make up the Society for Invertebrate Pathology (source: http://www.sipweb.org/)

Personally I am the most involved in the Microsporidia division, but have been attending some tremendously interesting talks across all of the divisions!

As my current postdoc work is not focused on pathogens, I presented a poster at this meeting on a side project that I did in 2013 where I surveyed local honey bees for Nosema apis and Nosema ceranae (microsporidia) in Mo’orea and Tahiti, French Polynesia. Below is a clip of my poster!

Slide1

Some of my favorite talks have ranged from discussing the ‘Pathobiome (discussed briefly in this linked article)‘ to Biological Control of Bed-Bugs using Beauveria bassiana (in video form here) to Nematode behavior and host searching (nematodes are so cool!), to the talks in the symposium featuring Ann Hajek and David Shapiro Ilan’s new book “Ecology of Invertebrate Diseases” with many of the great authors highlighting findings of their specific chapters (release date is November 2017!).  I also of course enjoyed hearing all of the famous microsporidian researchers discuss their work in the Microsporidian Symposium, including the work of four microsporidian researchers I hadn’t met in person prior to the meeting: Charles Vossbrinck and his work on the phylogenetics of microsporidia, Joe Maddox, John Henry (his work on the biological control of grasshoppers with Nosema locustae) and Louis Weiss’s work on human microsporidia (and of course on the similarities/differences to microsporidia in invertebrates). Here is the full meeting program from this year.

IMG_7541
Most of the participating Microsporidian researchers after our symposium

I also attended a workshop on RNAseq and Bioinformatics on Monday evening, and learned about the wonderful online GUI platform: Galaxy. This is a great platform to upload your RNAseq data and to do bioinformatics WITHOUT ANY CODING! WOW!

As the Student and Postdoctoral Chair for SIP, I was also responsible this year for organizing the youtube video contest (see all the winners here!), and the workshops and meetings for the students and postdocs. For the Student Workshop this year, we had a workshop on Science Communication- including how to work with the press and media, giving an elevator talk, and using social media to your advantage. (If you are interested- you can read more about some tips for science communication here). Then for the student business meeting, all of the students and postdocs (myself included) discussed what they would like to see in future SIP meetings (workshops on career opportunities, and scientific writing seemed to be the most popular requests!).

It was fun interacting with all of the students and postdocs, but definitely resulted in a busier conference than I’ve ever had before… it was difficult to get in my workouts- but nevertheless I succeeded with fitting some short workouts in and still have my ~ 10 year streak going (haven’t missed a day since Oct. 2007.. and that was because I was on a plane to New Zealand for almost 24 hours…)…

The other awesome part about this conference is that every year we spend half of a day going on group excursions. This year we went to the San Diego Zoo (and some folks did the Midway Excursion) – and it was a great chance to catch up with other researchers about both life and science!

In addition, every night we have social events ranging from a BBQ (this year there was no BBQ meat though which was funny- but we had fantastic tacos!), A Welcome Banquet and a Goodbye Banquet with Dancing (the last night is always the best!).

We also have a 5K run/walk every year- which I  usually attend but this year I stayed at a friend’s house a bit far away from the conference and couldn’t manage to get up early enough for that… ha (5:30am wake up I think not!).

And with that, I look forward to next year’s SIP conference in Australia (I’m trying to research potential fellowships for that as I will not have my current fellowship at this time next year).